cGMP regulates hydrogen peroxide accumulation in calcium-dependent salt resistance pathway in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> roots

3′,5′-cyclic guanosine monophosphate (cGMP) is an important second messenger in plants. In the present study, roles of cGMP in salt resistance in Arabidopsis roots were investigated. Arabidopsis roots were sensitive to 100 mM NaCl treatment, displaying a great increase in electrolyte leakage and Na⁺/K⁺ ratio and a decrease in gene expression of the plasma membrane (PM) H⁺-ATPase. However, application of exogenous 8Br-cGMP (an analog of cGMP), H₂O₂ or CaCl₂ alleviated the NaCl-induced injury by maintaining a lower Na⁺/K⁺ ratio and increasing the PM H⁺-ATPase gene expression. In addition, the inhibition of root elongation and seed germination under salt stress was removed by 8Br-cGMP. Further study indicated that 8Br-cGMP-induced higher NADPH levels for PM NADPH oxidase to generate H₂O₂ by regulating glucose-6-phosphate dehydrogenase (G6PDH) activity. The effect of 8Br-cGMP and H₂O₂ on ionic homeostasis was abolished when Ca²⁺ was eliminated by glycol-bis-(2-amino ethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA, a Ca²⁺ chelator) in Arabidopsis roots under salt stress. Taken together, cGMP could regulate H₂O₂ accumulation in salt stress, and Ca²⁺ was necessary in the cGMP-mediated signaling pathway. H₂O₂, as the downstream component of cGMP signaling pathway, stimulated PM H⁺-ATPase gene expression. Thus, ion homeostasis was modulated for salt tolerance.     

Rhizodeposition and C-partitioning of Lolium perenne in axenic culture affected by nitrogen supply and defoliation

This study investigated the effects of N-supply and partial defoliation on C-partitioning, root morphology and soluble rhizodeposition, for Lolium perenne grown in axenic sand culture systems percolated with nutrient solution. Plants were grown for 36 d in nutrient solutions with differing N concentrations (4 mM or 0.02 mM NH₄ ⁺NO₃ ^-), and effects of repeated defoliation to 4 cm were determined. The ‘low N’ supply reduced (P < 0.05) dry matter accumulation, with proportionately increased partitioning to the root systems. Root morphology was also altered at ‘low N’, with development of a finer root system, manifest as increased (P < 0.05) specific root length. Concurrent with these effects on growth of L. perenne, ‘low N’ increased (P < 0.05) exudation of C-compounds from roots on a per g root basis. Defoliation was found to increase exudation (P < 0.05) of soluble compounds for periods of 3-5 d following each cut, at both N-supply rates. The effects of N-supply and defoliation are of importance in understanding the coupling of plant productivity to nutrient cycling in soils with differing N availabilities and for grassland systems which are subject to grazing. This revised version was published online in June 2006 with corrections to the Cover Date.     

Exogenous ethylene influences flower opening of cut roses (<Emphasis Type="Italic">Rosa hybrida</Emphasis>) by regulating the genes encoding ethylene biosynthesis enzymes

The purpose of this paper is to investigate the differential responses of flower opening to ethylene in two cut rose cultivars, ‘Samantha’, whose opening process is promoted, and ‘Kardinal’, whose opening process is inhibited by ethylene. Ethylene production and 1-aminocyclopropane-1-carboxylate (ACC) synthase and oxidase activities were determined first. After ethylene treatment, ethylene production, ACC synthase (ACS) and ACC oxidase (ACO) activities in petals increased and peaked at the earlier stage (stage 3) in ‘Samantha’, and they were much more dramatically enhanced and peaked at the later stage (stage 4) in ‘Kardinal’ than control during vasing. cDNA fragments of three Rh-ACSs and one Rh-ACO genes were cloned and designated as Rh-ACS1, Rh-ACS2, Rh-ACS3 and Rh-ACO1 respectively. Northern blotting analysis revealed that, among three genes of ACS, ethylene-induced expression patterns of Rh-ACS3 gene corresponded to ACS activity and ethylene production in both cultivars. A more dramatic accumulation of Rh-ACS3 mRNA was induced by ethylene in ‘Kardinal’ than that of ‘Samantha’. As an ethylene action inhibitor, STS at concentration of 0.2 mmol/L generally inhibited the expression of Rh-ACSs and Rh-ACO in both cultivars, although it induced the expression of Rh-ACS3 transiently in ‘Kardinal’. Our results suggests that ‘Kardinal’ is more sensitive to ethylene than ‘Samantha’; and the changes of Rh-ACS3 expression caused by ethylene might be related to the acceleration of flower opening in ‘Samantha’ and the inhibition in ‘Kardinal’. Additional results indicated that three Rh-ACSs genes were differentially associated with flower opening and senescence as well as wounding.     

Selection of independent Ds transposon insertions in somatic tissue of potato by protoplast regeneration

Potato is an autotetraploid crop plant that is not very amenable to the deployment of transposon tagging for gene cloning and gene identification. After diploidisation it is possible to get potato genotypes that grow well, but they are self-incompatible. This prevents the production of selfed progeny that are normally used in gene tagging approaches to select for parental lines with the target gene to be tagged in a homozygous stage. We describe here an alternative selection method for directed transposon tagging for a gene of interest in a heterozygous background. Diploid potato plants with a Ds transposon linked to the desired gene of interest (the Phytophthora infestans R1 resistance locus) in a heterozygous stage were used for the development of this directed transposon tagging strategy. After crossing to a diploid Ac transposon-containing genotype, 22 ’interesting’ seedlings (R1Ds/r–; Ac/–) were selected that showed active Ds transposition as displayed by DNA blot hybridisation, empty donor site PCR and sequencing. Protoplast isolation and the use of the hygromycin gene as a cell-specific selection marker of Ds excision enabled the direct selection of Ds excision sectors in these highly chimaeric seedlings. This somatic selection of Ds transpositions and the regeneration through protoplasts resulted in the development of a large population of almost 2000 hygromycin-resistant plants. Southern blot analysis confirmed the insertion of Ds at independent positions in the genome. Every selected plant displayed independent Ds excisions and re-insertions due to the expression of the Ac transposase throughout development. This population, which is developed from seedlings with the desired R1 gene in a heterozygous stage, is directly useful for searching for transposon-tagged R1 mutants. In general, this approach for selecting for somatic transpositions is particularly suitable for the molecular isolation of genes in a heterozygous crop like potato. Received: 29 November 1999 / Accepted: 30 December 1999     

A GA-insensitive dwarf mutant of <Emphasis Type="Italic">Brassica napus</Emphasis> L. correlated with mutation in pyrimidine box in the promoter of <Emphasis Type="Italic">GID1</Emphasis>

A dwarf mutant from Brassica napus, namely NDF-1, which was derived from a high doubled haploid (DH) line ‘3529’(Brassica napus L.) of which seeds were jointly treated with chemical inducers and fast neutron bombardment, was revealed that dwarfism is under the control of a major gene(designated as ndf1) with a mainly additive effect and non-significant dominance effect. The germination and hypocotyls elongation response of dwarf mutants after exogenous GA and uniconazol application showed NDF-1 was a gibberellin insensitive dwarf. We cloned the Brassica napus GID1 gene, named BnGID1, and found it was the ortholog of AtGID1a. The sequence blasting of the BnGID1 genes from NDF-1 and wild type showed there was no mutant in the gene. But the quantitative RT-PCR analysis of GID1 EST pointed out the mutation was caused by the low-level expression of BnGID1 gene. After sequenced the BnGID1 gene’s upstream, we found three bases mutated in the pyrimidine box (P-box) of the BnGID1 promoter, which is linkage with the dwarf mutant.     

A chimeric <Emphasis Type="Italic">Rfo</Emphasis> gene generated by intergenic recombination cosegregates with the fertility restorer phenotype for cytoplasmic male sterility in radish

In this work, we have identified a chimeric pentatricopeptide repeat (PPR)-encoding gene cosegregating with the fertility restorer phenotype for cytoplasmic male sterility (CMS) in radish. We have constructed a CMS-Rf system consisting of sterile line ‘9802A2’, maintainer line ‘9802B2’ and restorer line ‘2007H’. F₂ segregating population analysis indicated that male fertility is restored by a single dominant gene in the CMS-Rf system described above. A PPR gene named Rfoc was found in the restorer line ‘2007H’. It cosegregated with the fertility restorer in the F₂ segregating population which is composed of 613 fertile plants and 187 sterile plants. The Rfoc gene encodes a predicted protein 687 amino acids in length, comprising 16 PPR domains and with a putative mitochondrial targeting signal. Sequence alignment showed that recombination between the 5′ region of Rfob (EU163282) and the 3′ region of PPR24 (AY285675) resulted in Rfoc, indicating a recent unequal crossing-over event between Rfo and PPR24 loci at a distance of 5.5 kb. The sterile line ‘9802A2’ contains the rfob gene. In the F₂ population, Rfoc and rfob were observed to fit a segregation ratio 1:2:1 showing that Rfoc was allelic to Rfo. Previously we have reported that a fertile line ‘2006H’, which carries the recessive rfob gene, is able to restore the male fertility of CMS line ‘9802A1’ (Wang et al. in Theor Appl Genet 117:313–320, 2008). However, here when conducting a cross between the fertile line ‘2006H’ and CMS line ‘9802A2, the resulting plants were male sterile, which shows that sterile line ‘9802A2’ possesses a different nuclear background compared to ‘9802A1’. Based on these results, the genetic model of fertility restoration for radish CMS is also discussed.     

Isolation,Characterization, and Expression Analysis of an <Emphasis Type="Italic">SKP1-</Emphasis>like Gene from ‘Shatian’ Pummelo (<Emphasis Type="Italic">Citrus grandis</Emphasis> Osbeck)

Plant s-phase kinase-associated protein 1 (SKP1) genes have diverse functions in plant developmental and physiological activities. Herein, we described a novel SKP1 gene, designated as CgSKP1, from ‘Shatian’ pummelo (Citrus grandis Osbeck). The cDNA sequence of CgSKP1 was 603 bp and contained an open reading frame of 477 bp. Genomic sequence of the CgSKP1 gene contained two exons and one intron. The predicted amino acid sequence of this gene is consisted of 158 amino acids with theoretical proteins size of 17.9 kDa. CgSKP1 had high identity with SKP1 genes from other plant species within two conserved region. Full-length cDNAs were also amplified and cloned from six citrus varieties, with 95% nucleotide identity and about 98% amino acid similarity among them. Gel blot analysis suggested that CgSKP1 existed as a single locus in the ‘Shatian’ pummelo genome. The expression of CgSKP1 was gradually increased during flower developmental stages in ‘Shatian’ pummelo. Moreover, expression analysis by RT-PCR, qRT-PCR and in situ hybridization of CgSKP1 showed that it was highly expressed in the leaf, petal, anther and ovary, but lowly in the style. These findings indicated that CgSKP1 was closely related to ‘Shatian’ pummelo flower development.     

Glutamate antibodies repress expression of <Emphasis Type="Italic">Dffb</Emphasis> gene in brain of rats in experimental Alzheimer’s disease

The rat model of Alzheimer’s disease including injection of neurotoxic fragment of β-amyloid protein Aβ_25–35 into giant-cell nuclei basalis of Meynert was used for experiments. We have investigated the influence of glutamate antibodies administered intranasally in a dose of 300 μg/kg after 1 h of the mentioned alteration on the level of expression of Dffb mRNA. Dffb gene codes caspase-dependent DNase, which participates in the internucleosomal fragmentation of genome DNA during apoptosis. On the third day after the injection of Aβ_25–35, we obtained a significant decrease in Dffb gene expression in the prefrontal cortex (37% decrease) and hippocampus (62% decrease) in the group of experimental animals compared to the control group. In the hypothalamus, there were no such differences. Seemingly, the repressing action of glutamate antibodies on the mRNA expression of the Dffb gene reflects the stabilization of processes that take place in the brain cells during experimental Alzheimer’s disease; meanwhile, the intensity of the apoptotic death of neurons and glial cells decreases.